Introducing a new breed of wine yeast: interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae

Abstract

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.

Figure 1. Genetic confirmation of cell hybridization by rDNA ITS PCR-RFLP.

Lane 1 100 bp ladder, lane 2 AWRI838, lane 3 NCYC2888, lane 4 DNA from both parents, lanes 5–9 Hybrids CxM1-5.

Figure 2. Sample sets of array-CGH data for parents and hybrid strain CxM1.

Within each panel of microarray data, each column contains the a-CGH data for a given strain while each row corresponds to a probe for a chromosomal location. The leftmost three panels show the data for probes to the S. cerevisiae genome, located on chromosome V (“YD’ followed by chromosome coordinate), XIV (‘YN”), and XVI (”YP”); the rightmost three panels show data for probes to various regions (contig “c” followed by contig number) of the S. mikatae genome. 838 is the S. cerevisiae parent strain, AWRI 1529 is the S. mikatae parent strain NCYC2888, and AWRI2526 is the hybrid strain CxM1. Red hybridisation intensities for a probe indicate the presence of that species’ genome region, while green hybridisation intensities indicate the absence of that species’ genome region. The reduced intensity of S. mikatae probes in the hybrid dataset indicates a reduced S. mikatae ploidy level relative to S. cerevisiae, within the hybrid genome.

Figure 3. Fluorescence flow cytometry analysis.

Top row left to right; Control ploidy strains BY4742 (haploid), BY4743 (diploid) and 53–7 (tetraploid). Middle row left to right; Parent strains AWRI838 and NCYC2888. Bottom row left to right; Hybrid strains CxM1 and CxM4.

Figure 4. Phenotypic assessment assay plates.

Top row plates left to right; YEPD at temperatures 22°C, 4°C and 37°C. Bottom row plates left to right; YEP 25% glucose, YEPD 14% ethanol. Strains are plated in columns at 10 fold serial dilutions from top to bottom; columns 1–5 CxM5-CxM1 in descending order, column 6 NCYC2888, column 7 AWRI838.

Figure 5. Grape juice fermentation profile of AWRI 838 and hybrid strains CxM1-CxM5.

Figure 5a. (top) Cell growth during fermentation as determined by Optical Density. Data points are presented with error bars. Figure 5b. (bottom) Sugar utilisation during fermentation as determined by Refractive Index. Data points are presented with error bars.

Figure 6. Genetic stability of S. cerevisiae x S. mikatae hybrids using rDNA ITS PCR-RFLP.

Top gel, CxM1 fermentation isolates and bottom gel, CxM4 fermentation isolates. Lane 1 100 bp ladder, lane 2 AWRI838, lane 3 NCYC2888, lane 4 DNA from both parents, lane 5, Hybrid, lanes 6–55 isolates 1–50. Arrow points to isolate with loss of S. mikatae rDNA.

Figure 7. Genetic stability of CxM1 fermentation isolates using chromosomal targeted PCR-RFLP.

First gel Chromosome XIV left arm, second gel Chromosome XIV right arm, third gel Chromosome XVI left arm and fourth gel Chromosome XVI right arm. Lane 1 100 bp ladder, lane 2 AWRI838, Lane 3 NCYC2888, lane 4 DNA from both parents, lane 5 Hybrid CxM1, lanes 6 to 55 isolates 1 to 50. Arrows point to isolates with altered chromosomal content.

Figure 8. Genetic stability of CxM4 fermentation isolates using chromosomal targeted PCR-RFLP.

First gel Chromosome XIV left arm, second gel Chromosome XIV right arm, third gel Chromosome XVI left arm and fourth gel Chromosome XVI right arm. Fifth gel Chromosome XII left arm, sixth gel Chromosome XII right arm, seventh gel Chromosome XIV left arm. Lane 1 100 bp ladder, lane 2 AWRI838, Lane 3 NCYC2888, lane 4 DNA from both parents, lane 5 Hybrid CxM4, lanes 6 to 55 isolates 1 to 50. Arrows point to isolates with altered chromosomal content.